- Email: [email protected]
A New Preservation Solution (SCOT 15) Improves the Islet Isolation Process From Pancreata of Non–Heart-Beating Donors: A Murine Model
A New Preservation Solution (SCOT 15) Improves the Islet Isolation Process From Pancreata of Non–Heart-Beating Donors: A Murine Model S. Giraud, T. Hauet, M. Eugene, G. Mauco, and B. Barrou ABSTRACT Introduction. Due to the organ shortage, there is increased use of organs harvested from non– heart-beating donors (NHBD). These organs have been subjected to a period of warm ischemia that is most deleterious to functional recovery. We have designed a new preservation solution, “Solution de Conservation des Organes et des Tissus” (SCOT 15; Macopharma, Tourcoing, France) which contains an extracellular ionic composition including PEG 20 kD (15 g/L) as a colloid. Methods. Our objective was to compare SCOT 15 with University of Wisconsin (UW) solution or islet culture medium CMRL 1066 ⫹ 1% of Bovine Serum Albumin (BSA), as the working and preservation solution for islet isolation from pancreata subjected to warm ischemia using a murine model. Results. Warm ischemia decreased the islet yield and cellular viability regardless of the preservation solution. Either when the pancreas was or was not subjected to warm ischemia, the best islet yield was obtained with SCOT 15 (P ⬍ .05 vs UW or CMRL 1066). The same results were observed for islet viability as assessed using the 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) test; namely, better viability with SCOT 15 as compared with UW or CMRL 1066 (P ⬍ .01). Conclusion. In a murine model SCOT 15 was a better preservation solution for islet isolation than UW solution or culture medium (CMRL 1066). SLET transplantation has become a feasible treatment option for patients with type 1 diabetes,1,2 but one of the major limitations is the decreased ratio of graft supply to demand. Currently, in France, the majority of pancreata are harvested from brain-dead donors; however, the recent launch of the non– heart-beating donor (NHBD) program has raised the question of the suitability of islets isolated from pancreata subjected to a warm ischemia period. Currently the most often used solutions are the University of Wisconsin (UW) solution and the culture medium CMRL 1066. We have designed a new preservation solution, “Solution de Conservation d’Organes et de Tissue” (SCOT 15; Macopharma, Tourcoing, France), which is approved for organ preservation.3 SCOT 15 has 2 main characteristics: (1) a low K⫹ concentration to avoid cell membrane depolarization and adenosine triphosphate (ATP) depletion, and (2) the presence of 15 g/L of polyethyleneglycol (PEG) 20 kd as colloid that has some immunoprotective effects as previously shown by our
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group.4,5 The beneficial effect of SCOT 15 has been established in models of isolated perfused pig kidneys and their autotransplantation6,7; it decreased the ischemia-reperfusion injuries. In addition, in our model of murine islet transplantation, SCOT 15 significantly improved the islet yield, pro-
From Institut National de la Santé et de la Recherche Médicale U927 (S.G., T.H., M.E., G.M., B.B.), Centre Hospitalier Universitaire, Poitiers, Faculté de Médecine, Université Pierre et Marie Curie, Paris VI (B.B.), and Service d’Urologie, GH Pitié-Salpétrière (B.B.), Paris, France. This work was supported by grants from Fondation pour la Recherche Médicale, Institut National de la Santé et de la Recherche Médicale (INSERM) and Centre Hospitalier Universitaire de Poitiers, Poitiers, France. Address reprint requests to Benoit Barrou, MD, PhD, Service d’Urologie, Unite de Transplantation Rénale et Pancréatique, Groupe Hospitalier Pitie-Salpétrière, 83 boulevard de l’Hôpital, 75013 Paris, France. E-mail: [email protected]
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.08.042
Transplantation Proceedings, 41, 3293–3295 (2009)
3293
3294
GIRAUD, HAUET, EUGENE ET AL Table 1. Human Versus Murine Warm Ischemia Time Equivalence Human heart beat ⁄ min ⫻ 30 min warm ischemia Murine heart beat ⁄ min Human O2 consumption ⫻ 30 min warm ischemia Murine O2 consumption
⫽ (70 ⫻ 30) ⁄ 600 ⫽ 3.5 min
⫽ (200 ⫻ 30) ⁄ 1680 ⫽ 3.5 min
Note: Calculations to find an equivalence between human and mice in warm ischemia time. According to this hypothesis, 30 minutes of no flow in humans is equivalent to 3.5 minutes in mice.
vided immunoprotection, and consequently increased islet allograft survival in the absence of recipient immunosuppression (P ⬍ .05). In addition, it achieved a decreased rate of primary nonfunction.5 The aim of our study was to determine whether SCOT 15 could improve the islet isolation process from pancreata subjected to a warm ischemia period, as is the case among NHBD. MATERIALS AND METHODS C3H (H-2k) mice obtained from Charles River France (L’arbresle, France) were used at 6 weeks of age. Mice were kept under specific pathogen-free conditions and manipulated according to European council directive 86/609/EEC. Islets were isolated as previously described5– 8 using stationary digestion with Liberase RI (Roche Diagnostics, Meylan, France) after in situ distension of the pancreas. Islets purified on a discontinuous Ficoll gradient were handpicked to obtain about 100% purity. Islets were isolated from C3H donor pancreata subjected to 3.5 minutes of warm ischemia or not (3.5 minutes in mice is equivalent to 30 minutes in humans, (Table 1). Isolated islets were preserved using 3 solutions: (1) UW solution as the standard preservation solution; (2) CMRL 1066 ⫹ 1% bovine serum albumin (BSA), as used in clinical islet isolation, or (3) SCOT 15. After isolation, islets were counted according to the islet equivalent (IEQ) method9 to determine yield (n ⫽ 5 for each condition). Cellular viability was determined using the 3,(4,5diméthylthiazol-2-yl)-2,5-diphényl tétrazolium bromide (MTT) test, based on the cleavage of the yellow tetrazolium salt MTT to a purple formazan crystal by metabolically active cells. The formazan
was then solubilized, and its concentration determined by optical density at 570 nm (n ⫽ 3 for each condition). All data are expressed as mean values ⫾ SEM. Statistical analyses were performed using analysis of variance (ANOVA) followed by Kruskal-Wallis test with P ⬍ .05 considered significant.
RESULTS
Islet yield from pancreata after warm ischemia was better with SCOT 15 solution (328 ⫾ 23 IEQ) compared with CMRL (256 ⫾ 8 IEQ; P ⬍ .05) or UW (98 ⫾ 10 IEQ; P ⬍ .01) (Figure 1). Similar results were observed with pancreata harvested without warm ischemia; the islet yield was better with SCOT 15 (593 ⫾ 8 IEQ) as compared with CMRL (494 ⫾ 16 IEQ; P ⬍ .05) or UW solution (356 ⫾ 32 IEQ; P ⬍ .01). In terms of cellular viability, islets from the warm ischemia pancreata were better preserved with SCOT 15 solution (0.146 ⫾ 0.02 absorbance) than UW (0.093 ⫾ 0.01 absorbance; P ⬍ .01) or CMRL (0.130 ⫾ 0.01 absorbance; P ⬍ .01), as islet viability from pancreata without warm ischemia were better with SCOT 15 (0.219 ⫾ 0.01 absorbance) than UW (0.138 ⫾ 0.02 absorbance; P ⬍ .01) or CMRL (0.136 ⫾ 0.01 absorbance; P ⬍ .01). DISCUSSION
This study showed that warm ischemia decreased the islet yield and cellular viability regardless of the solution. Concordant observations have been reported by other workers.10 –12
Fig 1. Yield and viability of islet preserved with different solutions after warm ischemia or not. (A) Islet yield expressed in IEQ/pancreas obtained after isolation in different conditions (n ⫽ 5 for each condition). (B) Cellular viability of islet 1 hour after isolation in different conditions (n ⫽ 3 for each condition). In white □, islet obtained without warm ischemia; in black stripe , islet obtained after 3.5 minutes of warm ischemia. *P ⬍ .05 (vs UW without warm ischemia). P ⬍ .05 (vs UW ⫹ warm ischemia). #P ⬍ .05 (warm ischemia vs not).
NEW PRESERVATION SOLUTION
The use of SCOT 15 improved both the islet yield and the viability when compared with other solutions. These findings could make it possible to use pancreata harvested from NHBD as suggested by other investigators.13 REFERENCES 1. Merani S, Shapiro AM: Current status of pancreatic islet transplantation. Clin Sci (Lond) 110:611, 2006 2. Shapiro AM, Ricordi C, Hering BJ, et al: International trial of the Edmonton protocol for islet transplantation. N Engl J Med 355:1318, 2006 3. Badet L, Eugène M, Hauet T, et al: L’utilisation des liquides de conservations en transplantation rénale. Progrès Urol 16:25, 2006 4. Eugene M: Polyethyleneglycols and immunocamouflage of the cells tissues and organs for transplantation. Cell Mol Biol 50:209, 2004 5. Giraud S, Claire B, Eugene M, et al: A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation. Transplantation 83:1397, 2007 Erratum in: Transplantation 84:561, 2007 6. Hauet T, Goujon JM, Baumert H, et al: Polyethylene glycol reduces the inflammatory injury due to cold ischemia/reperfusion in autotransplanted pig kidneys. Kidney Int 62:654, 2002
3295 7. Eugene M, Hauet T, Mothes D, et al: Beneficial effects of a low potassium and polyethylene glycol solution on renal function and structure during 48-hour cold storage preservation. Transplant Proc 29:2360, 1997 8. Giraud S, Barrou B, Sebillaud S, et al: Transient depletion of dividing T lymphocytes in mice induces the emergence of regulatory T cells and dominant tolerance to islet allografts. Am J Transplant 8:942, 2008 9. Ricordi C, Gray DW, Hering BJ, et al: Islet isolation assessment in man and large animals. Acta Diabetol Lat 27:185, 1990 10. Brandhorst D, Iken M, Bretzel RG, et al: Pancreas storage in oxygenated perfluorodecalin does not restore post-transplant function of isolated pig islets pre-damaged by warm ischemia. Xenotransplantation 13:465, 2006 11. Tanaka T, Suzuki Y, Tanioka Y, et al: Possibility of islet transplantation from a nonheartbeating donor pancreas resuscitated by the two-layer method. Transplantation 80:738, 2005 12. Solowiej E, Solowiej J, Kasprzycka-Guttman T, et al: Application of sulforaphane— does it lead to improvement of islet graft survival after warm and/or cold ischemia. Ann Transplant 9:68, 2004 13. Clayton HA, Swift WM, Turner JM, et al: Non-heart-beating organ donors: a potential source of islets for transplantation? Transplantation 69:2094, 2000
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group.4,5 The beneficial effect of SCOT 15 has been established in models of isolated perfused pig kidneys and their autotransplantation6,7; it decreased the ischemia-reperfusion injuries. In addition, in our model of murine islet transplantation, SCOT 15 significantly improved the islet yield, pro-
From Institut National de la Santé et de la Recherche Médicale U927 (S.G., T.H., M.E., G.M., B.B.), Centre Hospitalier Universitaire, Poitiers, Faculté de Médecine, Université Pierre et Marie Curie, Paris VI (B.B.), and Service d’Urologie, GH Pitié-Salpétrière (B.B.), Paris, France. This work was supported by grants from Fondation pour la Recherche Médicale, Institut National de la Santé et de la Recherche Médicale (INSERM) and Centre Hospitalier Universitaire de Poitiers, Poitiers, France. Address reprint requests to Benoit Barrou, MD, PhD, Service d’Urologie, Unite de Transplantation Rénale et Pancréatique, Groupe Hospitalier Pitie-Salpétrière, 83 boulevard de l’Hôpital, 75013 Paris, France. E-mail: [email protected]
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.08.042
Transplantation Proceedings, 41, 3293–3295 (2009)
3293
3294
GIRAUD, HAUET, EUGENE ET AL Table 1. Human Versus Murine Warm Ischemia Time Equivalence Human heart beat ⁄ min ⫻ 30 min warm ischemia Murine heart beat ⁄ min Human O2 consumption ⫻ 30 min warm ischemia Murine O2 consumption
⫽ (70 ⫻ 30) ⁄ 600 ⫽ 3.5 min
⫽ (200 ⫻ 30) ⁄ 1680 ⫽ 3.5 min
Note: Calculations to find an equivalence between human and mice in warm ischemia time. According to this hypothesis, 30 minutes of no flow in humans is equivalent to 3.5 minutes in mice.
vided immunoprotection, and consequently increased islet allograft survival in the absence of recipient immunosuppression (P ⬍ .05). In addition, it achieved a decreased rate of primary nonfunction.5 The aim of our study was to determine whether SCOT 15 could improve the islet isolation process from pancreata subjected to a warm ischemia period, as is the case among NHBD. MATERIALS AND METHODS C3H (H-2k) mice obtained from Charles River France (L’arbresle, France) were used at 6 weeks of age. Mice were kept under specific pathogen-free conditions and manipulated according to European council directive 86/609/EEC. Islets were isolated as previously described5– 8 using stationary digestion with Liberase RI (Roche Diagnostics, Meylan, France) after in situ distension of the pancreas. Islets purified on a discontinuous Ficoll gradient were handpicked to obtain about 100% purity. Islets were isolated from C3H donor pancreata subjected to 3.5 minutes of warm ischemia or not (3.5 minutes in mice is equivalent to 30 minutes in humans, (Table 1). Isolated islets were preserved using 3 solutions: (1) UW solution as the standard preservation solution; (2) CMRL 1066 ⫹ 1% bovine serum albumin (BSA), as used in clinical islet isolation, or (3) SCOT 15. After isolation, islets were counted according to the islet equivalent (IEQ) method9 to determine yield (n ⫽ 5 for each condition). Cellular viability was determined using the 3,(4,5diméthylthiazol-2-yl)-2,5-diphényl tétrazolium bromide (MTT) test, based on the cleavage of the yellow tetrazolium salt MTT to a purple formazan crystal by metabolically active cells. The formazan
was then solubilized, and its concentration determined by optical density at 570 nm (n ⫽ 3 for each condition). All data are expressed as mean values ⫾ SEM. Statistical analyses were performed using analysis of variance (ANOVA) followed by Kruskal-Wallis test with P ⬍ .05 considered significant.
RESULTS
Islet yield from pancreata after warm ischemia was better with SCOT 15 solution (328 ⫾ 23 IEQ) compared with CMRL (256 ⫾ 8 IEQ; P ⬍ .05) or UW (98 ⫾ 10 IEQ; P ⬍ .01) (Figure 1). Similar results were observed with pancreata harvested without warm ischemia; the islet yield was better with SCOT 15 (593 ⫾ 8 IEQ) as compared with CMRL (494 ⫾ 16 IEQ; P ⬍ .05) or UW solution (356 ⫾ 32 IEQ; P ⬍ .01). In terms of cellular viability, islets from the warm ischemia pancreata were better preserved with SCOT 15 solution (0.146 ⫾ 0.02 absorbance) than UW (0.093 ⫾ 0.01 absorbance; P ⬍ .01) or CMRL (0.130 ⫾ 0.01 absorbance; P ⬍ .01), as islet viability from pancreata without warm ischemia were better with SCOT 15 (0.219 ⫾ 0.01 absorbance) than UW (0.138 ⫾ 0.02 absorbance; P ⬍ .01) or CMRL (0.136 ⫾ 0.01 absorbance; P ⬍ .01). DISCUSSION
This study showed that warm ischemia decreased the islet yield and cellular viability regardless of the solution. Concordant observations have been reported by other workers.10 –12
Fig 1. Yield and viability of islet preserved with different solutions after warm ischemia or not. (A) Islet yield expressed in IEQ/pancreas obtained after isolation in different conditions (n ⫽ 5 for each condition). (B) Cellular viability of islet 1 hour after isolation in different conditions (n ⫽ 3 for each condition). In white □, islet obtained without warm ischemia; in black stripe , islet obtained after 3.5 minutes of warm ischemia. *P ⬍ .05 (vs UW without warm ischemia). P ⬍ .05 (vs UW ⫹ warm ischemia). #P ⬍ .05 (warm ischemia vs not).
NEW PRESERVATION SOLUTION
The use of SCOT 15 improved both the islet yield and the viability when compared with other solutions. These findings could make it possible to use pancreata harvested from NHBD as suggested by other investigators.13 REFERENCES 1. Merani S, Shapiro AM: Current status of pancreatic islet transplantation. Clin Sci (Lond) 110:611, 2006 2. Shapiro AM, Ricordi C, Hering BJ, et al: International trial of the Edmonton protocol for islet transplantation. N Engl J Med 355:1318, 2006 3. Badet L, Eugène M, Hauet T, et al: L’utilisation des liquides de conservations en transplantation rénale. Progrès Urol 16:25, 2006 4. Eugene M: Polyethyleneglycols and immunocamouflage of the cells tissues and organs for transplantation. Cell Mol Biol 50:209, 2004 5. Giraud S, Claire B, Eugene M, et al: A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation. Transplantation 83:1397, 2007 Erratum in: Transplantation 84:561, 2007 6. Hauet T, Goujon JM, Baumert H, et al: Polyethylene glycol reduces the inflammatory injury due to cold ischemia/reperfusion in autotransplanted pig kidneys. Kidney Int 62:654, 2002
3295 7. Eugene M, Hauet T, Mothes D, et al: Beneficial effects of a low potassium and polyethylene glycol solution on renal function and structure during 48-hour cold storage preservation. Transplant Proc 29:2360, 1997 8. Giraud S, Barrou B, Sebillaud S, et al: Transient depletion of dividing T lymphocytes in mice induces the emergence of regulatory T cells and dominant tolerance to islet allografts. Am J Transplant 8:942, 2008 9. Ricordi C, Gray DW, Hering BJ, et al: Islet isolation assessment in man and large animals. Acta Diabetol Lat 27:185, 1990 10. Brandhorst D, Iken M, Bretzel RG, et al: Pancreas storage in oxygenated perfluorodecalin does not restore post-transplant function of isolated pig islets pre-damaged by warm ischemia. Xenotransplantation 13:465, 2006 11. Tanaka T, Suzuki Y, Tanioka Y, et al: Possibility of islet transplantation from a nonheartbeating donor pancreas resuscitated by the two-layer method. Transplantation 80:738, 2005 12. Solowiej E, Solowiej J, Kasprzycka-Guttman T, et al: Application of sulforaphane— does it lead to improvement of islet graft survival after warm and/or cold ischemia. Ann Transplant 9:68, 2004 13. Clayton HA, Swift WM, Turner JM, et al: Non-heart-beating organ donors: a potential source of islets for transplantation? Transplantation 69:2094, 2000